Powerful genetic tools have made the zebrafish a promising model system. Currently available technologies include methods for forward genetics, reverse genetics and transgenesis. In contrast, however, a method for targeted manipulation of the zebrafish genome is lacking. Advances in the field of mouse genetics have shown that such a methodology is essential for the detailed and sophisticated analysis of gene function. We propose to develop and test tools for efficient genome manipulation in zebrafish. Building on homologous recombination technology in Drosophila, we will test if double-strand breaks introduced in vivo can lead to homologous recombination events in zebrafish. A vasa-GFP gene trap vector flanked by I-Scel sites and ioxP sites will be excised by Cre-mediated recombination, and i-Scel-mediated cleavage will result in double-strand breaks. Based on studies in Drosophila, we hypothesize that this DNA can now target the endogenous locus. We will test both positive and positive/negative selection protocols. Based on the results of vasa targeting, we will then target the no tail gene. [unreadable] [unreadable]